This project will test the hypothesis that supplementation of a DNA vaccine with recombinant HIV envelope protein plus adjuvant will induce strong antiviral humoral as well as cellular immune responses in both mucosal and systemic compartments if simultaneously administered by both the intradermal and nasal routes. Specific Aim I is to confirm that the parenteral adjuvant QS-21 can also be used to adjuvant immune responses to nasally administered DNA vaccines or soluble protein, and to establish an effective nasal QS-21 dosage for these vaccine preparations. Groups of female rhesus macaques will be nasally vaccinated with 10 or 25 pg QS-21 plus rgp41-hemagglutinin fusion protein (gp41HA) or DNA encoding noninfectious SHIV89.6P particles (SHIV DNA). HIV- and SIV-specific antibodies induced in sera and secretions will be quantitated by ELISA. T helper cell (Th) and cytotoxic T lymphocyte (CTL) responses in the periphery, rectal mucosa, and the cervical/vaginal mucosa will be evaluated by testing isolated mononuclear cells for antigen-specific lymphoproliferation, IFN-( secretion, and intracellular IFN-gamma production using the [3H] thymidine uptake assay, ELISPOT assay, and flow cytometry. The QS-21 dose found to produce greatest immune responses would be used for subsequent nasal vaccinations. Specific AIM II is to use QS-21, SHIV DNA, and gp41HA in nonhuman primates to identify an optimal, practical DNA/protein vaccination protocol for induction of humoral and cellular immune responses in the systemic compartment, rectal mucosa, and genital tract mucosa. We will first confirm that SHIV DNA plus gp41HA and QS-21 can be administered as one formulation without loss of immunogenicity. Ultimately, a vaccination strategy involving simultaneous nasal/intradermal administration of SHIV DNA, gp41HA, and QS-21 followed by simultaneous nasal/intradermal boosting with recombinant modified vaccinia ankara virus-expressing SHIV antigens will be tested for immunogenicity, and protective efficacy after rectal SHIV89.6P challenge. In all of these studies, the methods described above in AIM I will be used to analyze antibody and T cell responses, including those in the mucosa. Specific Aim III is to refine and optimize an HIV/epithelial cell-binding assay that could be used to test secretions of vaccine recipients for antibodies that block HIV attachment to epithelial cells. Secretions collected from animals vaccinated above will be analyzed in this assay to determine if the gp41HA vaccine antigen is capable of inducing mucosal antibodies that could potentially prevent entry of HIV into the host. This study is expected to significantly further our knowledge concerning adjuvants for DNA vaccines as well as the nasal vaccination route, the immunogenicity of potential HIM vaccine subunit components, methods that could be used for evaluating secretions for transmission-preventing mucosal antibodies, and the design of vaccination protocols that are most likely to induce protective immunity to HIV.